Oral squamous cell carcinoma is the 1st most common malignancy in oral and maxillofacial area. HPV 16 has beenstrongly linked to progression of cervical carcinoma. E6 and E7 as a small DNA virus encoding two major oncoproteinsof HPV 16 can act together to produce efficient immortalization of primary human epithelial cells. Thus it is importantto pursue the development of Immortalized human oral keratinocyte(IHOK) culture model which could be related to thepathogenesis of oral squamous cell carcinoma. If we establish IHOK transfected by E6/E7 genes, IHOK will be acceptedas a model system for HPV-linked oral carcinogenesis. The purpose of this study were to culture primary normal humanoral keratinocyte(NHOK), and to establish IHOK for studying oral carcinogenesis in the future. NHOK was primarilycultured under normal culture condition, and transformed into IHOK by transfection of E6/E7 genes. After 100 passagesdepend on Ca++ condition, cultured IHOK was confirmed by growth curve, cornified cell envelope measurement,TGase 1activity, mRNA detection, tumorogenecity and anchorage independence assay. After 100 passages, cultured IHOKshowed most basal cell and monolayer of polyhedral cells under 0.15mM Ca++, and small area of stratification andflattened epithelial cells with irregular border under 1.2mM Ca++. The cultured IHOK showed relatively resistantgrowth under high calcium condition. The E6/E7 mRNA was detected in cultured IHOK by RT-PCR. During the terminaldifferentiation in cultured IHOK, increased insoluble cornified cell envelope formation was accompanied with inductionof TGase 1 activity. But the cultured IHOK showed less CEM and TGase 1 activity than those of cultured NHOK.Cultured IHOK showed non-tumorogenecity, but slight anchorage independence. We had developed a technique totransform NHOK into IHOK by transfection of E6/E7 genes. Cultured IHOK was established as intermediate stage cellto study the pathogenesis of human oral squamous cell carcinoma.

Amino acid transporters are essential for the growth and proliferation in all living cells. Among the amino acidtransporters, the system L amino acid transporters are the major nutrient transport system responsible for theNa+-independent transport of neutral amino acids including several essential amino acids. The L-type amino acidtransporter 1 (LAT1) is over-expressed to support cell growth in malignant tumors. The double stranded RNA-mediatedRNA interference (RNAi) analysis can be in a wide variety of eukaryotes to induce the sequence-specific inhibition ofgene expression. In this study, we examined the effect of LAT1 short interfering RNA (siRNA) on cell growth usingsiRNA of LAT1 in the KB human oral squamous cell carcinoma. In the RT-PCR analysis and western blot analysis, thesiRNA of LAT1 inhibited expressions of LAT1 mRNA and protein. The uptake of [14C]L-leucine was inhibited by siRNAof LAT1. In the MTT assay, the siRNA of LAT1 inhibited the growth of the KB cells in the time-dependent manner,indicating that the growth inhibition of KB cell by the siRNA of LAT1 is induced by the blocking of neutral amino acidtransport mediated by LAT1. These results suggest that the transport of neutral amino acids including several essentialamino acids into the KB human oral squamous cell carcinoma is mediated mainly by LAT1. Further, the LAT1 would bea new target for the inhibition of cancer cell growth.